Neuropilin antisense oligonucleotide sequences and methods of using same to modulate cell growth

Abstract

This invention relates to oligonucleotides complementary to the neuropilin genes which modulate tumor cell growth and angiogenesis in mammals. This invention is also related to methods of using such compounds in inhibiting the growth of tumor cells and angiogenesis in mammals. This invention also relates to pharmaceutical compositions comprising a pharmaceutically acceptable excipient and an effective amount of a compound of this invention.

1. An antisense oligonucleotide from about 20 to 100 nucleotides in length comprising a sequence complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33 and said antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12, and wherein said antisense oligonucleotide specifically binds to the nucleic acid comprising the sequence of said mRNA and inhibits neuropilin expression in the human and inhibits tumor cell growth in a human. 
     
     
       2. A vector comprising a sequence encoding an antisense oligonucleotide from about 20 to 100 nucleotides in length, said antisense oligonucleotide comprising a sequence complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33 and said antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12, and wherein said antisense oligonucleotide specifically binds to the nucleic acid comprising the sequence of said mRNA and inhibits neuropilin expression in the human and inhibits tumor cell growth in the human. 
     
     
       3. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and an effective amount of an antisense oligonucleotide from about 20 to 100 nucleotides in length comprising a sequence complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33 and said antisense oligonucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12, and wherein said antisense oligonucleotide specifically binds to a nucleic acid comprising the sequence of said mRNA and inhibits neuropilin expression in a human and inhibits tumor cell growth in a human. 
     
     
       4. A method for inhibiting the growth of a human tumor comprising, administering to a human having the tumor an effective amount of an antisense oligonucleotide from about 20 to 50 nucleotides in length complementary to a human neuropilin mRNA under conditions such that the antisense oligonucleotide inhibits the growth of the tumor, wherein said mRNA has a sequence as set forth in SEQ ID NO:33, said tumor is derived from a carcinoma, and said antisense oligonucleotide specifically binds to a nucleic acid comprising the sequence of said mRNA. 
     
     
       5. The method according to  claim 4  further comprising the step of administering to the human a chemotherapeutic agent. 
     
     
       6. The method according to  claim 4  wherein the antisense oligonucleotide is from 20 to 50 nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       7. The method according to  claim 4  wherein the antisense oligonucleotide is nuclease resistant. 
     
     
       8. A method of inhibiting the growth of human cancer cells comprising, contacting said cancer cells in vitro with an effective amount of an antisense oligonucleotide from about 20 to 50 nucleotides in length complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33, under conditions such that the antisense oligonucleotide inhibits the growth of the cancer cells. 
     
     
       9. The method according to  claim 8 , wherein the antisense oligonucleotide is from 20 to 50 nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       10. The method according to  claim 8  wherein the antisense oligonucleotide is nuclease resistant. 
     
     
       11. The method according to  claim 4 , comprising administering said antisense oligonucleotide by infusion. 
     
     
       12. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       13. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide is a peptide nucleic acid. 
     
     
       14. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide comprises a morpholino backbone structure. 
     
     
       15. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide comprises at least one modified base selected from the group consisting of xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine, 8-halo guanines, 8-amino guanine, 8-thiol guanine, 8-thioalkyl guanines, 8-hydroxyl guanine, 5-trifluoromethyl uracil and 5-trifluoro cytosine. 
     
     
       16. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide comprises one or more modified intemucleotide linkages in the phosphate backbone selected from the group consisting of methyl phosphonate, phosphorothioate, phosphorodithioate and phosphotriester intemucleotide linkages. 
     
     
       17. The antisense oligonucleotide according to  claim 1 , wherein the antisense oligonucleotide comprises one or more phosphorothioate intemucleotide linkages. 
     
     
       18. The antisense oligonucleotide according to  claim 1 , wherein the antisense oligonucleotide comprises one or more alkyl, cycloalkyl or heterocyclic intersugar linkages. 
     
     
       19. The antisense oligonucleotide according to  claim 1 , wherein the antisense oligonucleotide comprises at least one nucleotide that is a 2′-O-substituted ribonucleotide. 
     
     
       20. The antisense oligonucleotide according to  claim 1 , wherein said antisense oligonucleotide is nuclease resistant. 
     
     
       21. The vector according to  claim 2 , wherein said antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       22. The pharmaceutical composition according to  claim 3  wherein said antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       23. The method according to  claim 4 , wherein said tumor is a cancer selected from the group consisting of melanoma, colon cancer, lung cancer, prostate cancer, pancreatic cancer and breast cancer. 
     
     
       24. A method of inhibiting colon cancer growth comprising, administering to a human having a colon cancer an effective amount of an antisense oligonucleotide from about 20 to 50 nucleotides in length complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33, and wherein said antisense oligonucleotide inhibits the growth of the colon cancer in the human. 
     
     
       25. The method according to  claim 24  further comprising the step of administering to the human a chemotherapeutic agent. 
     
     
       26. The method according to  claim 24 , wherein the antisense oligonucleotide is nuclease resistant. 
     
     
       27. The method according to  claim 24 , wherein the antisense oligonucleotide is from 20 to 50 nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       28. The method according to  claim 24 , wherein the antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       29. A method of inhibiting metastasis of a melanoma comprising, administering to a human having a melanoma an effective amount of an antisense oligonucleotide from about 20 nucleotides to 50 nucleotides in length complementary to a human neuropilin mRNA, wherein said mRNA has a sequence as set forth in SEQ ID NO:33, and wherein said oligonucleotide inhibits the metastasis of the melanoma in the human. 
     
     
       30. The method according to  claim 29 , further comprising the step of administering to the human a chemotherapeutic agent. 
     
     
       31. The method according to  claim 29 , wherein the oligonucleotide is nuclease resistant. 
     
     
       32. The method according to  claim 29 , wherein the oligonucleotide is from 20 to 50 nucleotides in length and comprises a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       33. The method according to  claim 29 , wherein the oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       34. The method according to  claim 24 , comprising administering said antisense oligonucleotide by infusion. 
     
     
       35. The method according to  claim 29 , comprising administering said antisense oligonucleotide by infusion. 
     
     
       36. The method according to  claim 4 , wherein the antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       37. The method according to  claim 8 , wherein the antisense oligonucleotide consists of a sequence selected from the group consisting of SEQ ID NOs:1, 2, 3, 5, 6, 8, 9, 10, 11 and 12. 
     
     
       38. The antisense oligonucleotide according to  claim 1 , wherein the antisense oligonucleotide is from about 20 to 50 nucleotides in length. 
     
     
       39. The vector according to  claim 2 , wherein the antisense oligonucleotide is from about 20 to 50 nucleotides in length. 
     
     
       40. The pharmaceutical composition according to  claim 3 , wherein the antisense oligonucleotide is from about 20 to 50 nucleotides in length.